In article <01HNP6F12GKI00PF24 at Post-Office.UH.EDU>
CPedemonte at UH.EDU (carlos h. pedemonte) writes:
> Path: news.uh.edu!swrinde!sgiblab!sgigate.sgi.com!olivea!biosci!UH.EDU!CPedemonte
> From: CPedemonte at UH.EDU (carlos h. pedemonte)
> Newsgroups: bionet.molbio.methds-reagnts
> Subject: Is it Taq or the primer responsible for the mistake?
> Date: 3 Mar 1995 10:22:41 -0800
> Organization: BIOSCI International Newsgroups for Molecular Biology
> Lines: 44
> Sender: daemon at net.bio.net> Distribution: world
> Message-ID: <01HNP6F12GKI00PF24 at Post-Office.UH.EDU>
> NNTP-Posting-Host: net.bio.net
>>>> We have sequenced part of a plasmid and found a missing base. Since we
> have used PCR with Taq polymerase to produce this part of the plasmid, we
> want to identify what have caused this deletion. We think that the problem
> is in the custom-made primer. But we would like to hear your opinion, to
> be sure that our conclusion is correct.
>> We used a custom-made primer to produce a fragment by PCR (with Taq
> polymerase). The fragment and the vector were cut with restriction enzymes
> and ligated. DH5-alpha competent cells were used for transfection.
> Colonies were screened by PCR. Two colonies were positive for the presence
> of the fragment. These colonies were picked from the plate to do sequencing
> (Sequenase PCR with Taq polymerase). In both sequences one base was
> missing; this base corresponded to the custom-made primer (not to a sector
> that was amplified by PCR). The sequence of one of the colonies was
> repeated. Again, the same base was missing.
>> OUR REASONING: The mistake can be produced by either i) Taq polymerase, or
> ii) the primer has a missing base. Taq polymerase could only have produced
> a mistake during the sequencing. THE MISTAKE CANNOT HAVE BEEN PRODUCED
> DURING THE PREPARATION OF THE FRAGMENT BECAUSE THE MISSING BASE IS IN A
> SECTOR THAT CORRESPOND TO THE PRIMER. Since it is very unlikely that Taq
> would produce three times the same mistake, we concluded that the primer is
> wrong. The mistake was done during the preparation of the primer.
>> 1) IS OUR CONCLUSION CORRECT?
>> 2) Can oligonucleotide synthesizers produce primers that are wrong?
>> Thank you for your help.
>> Best, carlos
>> ______________________________________________________________________________
> Carlos H. Pedemonte Telephone: 713-743-1211 (office)
> Dept. of Pharmacology 713-743-1228 (lab)
> University of Houston Fax: 713-743-1229
>> Houston, TX 77204-5515 e-mail: pedemonte at jetson.uh.edu> ____________________________________________________________________________
> __
>>
I agree with your reasoning. Did you receive the computer printout from
the company for your oligo? I have found on at least 2 occasions (out
of a few hundred oligos) that the company typed in my sequence
incorrectly. These were both changes and I found out by carefully
comparing the printout they sent me with the sequence I sent them.
So the simplest explanation would be a type.
Alternately there are possible reasons for a machine failure, such as
they changed reagents during your run, there was an air bubble in the
line, etc etc. So it is possible and not all oligos are perfect.
Michael Benedik benedik at uh.edu
Biochemical Sciences
University of Houston
Houston, TX 77204-5934